Methods for enhanced root nodulation in legumes

ABSTRACT

Disclosed herein are methods of increasing, enhancing, or accelerating root nodulation in a plant, accelerating growth of nitrogen fixing bacteria in nodules of a plant, increasing protein content in a plant, increasing yield of a plant, improving water retention of a plant, or reducing water use of a plant, the method comprising identifying a plant in need of root nodulation, and applying to the plant a composition comprising a protein component comprising yeast stress proteins resulting from subjecting a mixture obtained from the yeast fermentation to stress.

RELATED APPLICATION

The present application claims priority to the U.S. ProvisionalApplication Ser. No. 61/399,095, filed Jul. 7, 2010, and entitled“Methods for Enhanced Root Nodulation in Legumes,” the entire disclosureof which is incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to enhancement of plant growth and cropyield by applying compositions of fermentation liquids and surfactantthat improve root nodulation by rhizobacteria, e.g., Rhizobium,Bradyrhizobium, Sinorhizobium, etc.

BACKGROUND OF THE DISCLOSURE

Legumes are plants, such as alfalfa, clover, peas, beans, lentils,lupins, mesquite, carob, soy, peanuts, locust trees (Gleditsia orRobinia), wisteria, and the Kentucky coffeetree (Gymnocladus dioicus),that form a symbiotic relationship between their roots and bacteria,specifically of the family Rhizobiaceae. The bacteria penetrate theplant root hairs, and then induce the formation of nodules. The plantprovides the bacteria both sustenance and an energy source in the formof adenosine triphosphate (ATP) that is generated by photosynthesis. Inreturn, the bacteria are able to fix elemental nitrogen from theatmosphere into ammonia, a usable form of nitrogen that is digestible byplants thereby providing a rich nitrogen source to the plant. Thisprocess is called nitrogen fixation. Nitrogen is the nutrient that ismost frequently a limiting item to the growth of green plants andoptimizing its application is a key to optimizing plant yield. The termyield will be referred to as the crop which is being grown, be it peas,soybeans, or other legume.

Legumes are generally higher in protein content than other plantfamilies due to the availability of nitrogen from nitrogen fixation. Thehigh protein content makes legumes one of the most important food cropsfor both human consumption and animal feed. Further, legumes are used incrop rotation practice to increase the nitrogen content of soils,through nitrogen fixation, for future growth seasons and to reduce theamount of fertilizer that needs to be applied. This has cost benefits tothe grower and can reduce nitrogen runoff.

Each plant species requires a particular strain of Rhizobia species fornodulation to form. Native rhizobial populations are not typicallyoptimized for a particular plant species unless the crop grownpreviously was the specific legume to be planted. To optimize theeffectiveness of nodule formation, appropriate species of Rhizobia canbe inoculated into a crop. There are three methods of inoculation, eachwith its own advantages and limitations; solid, liquid and freeze-dried.Solid peat-based inoculants can be applied to seed or directly to thesoil. Liquid inoculants are mixed with water and applied to the seedfurrow at the time of planting. To maintain viability of the livebacteria, liquid inoculants must be kept frozen or refrigerated whenstored and during shipment. The handling requirements increase costs andfurther limit their availability through standard distribution.Seed-applied inoculants are the most commonly used and precautions inhandling need to be employed to preserve the live bacteria. A keylimitation with inoculating legumes to maximize yield is that even underthe best storage conditions, rhizobial populations will decline overtime.

The nitrogen fixation process is a transfer of electrons by oxidizinghydrogen and reducing elemental nitrogen to form ammonia. The reductionof nitrogen is an energy intensive process, and, to fix elementalnitrogen, the rhizobial bacteria gets its energy from the plant that itinfects. The chemical process involves a two-part enzyme system known asnitrogenase. The system contains iron and is highly susceptible to beinginactivated in the presence of oxygen. This is not a problem withanaerobic bacteria. However, nitrogen fixing aerobic bacteria, such asRhizobium in the soil can overcome the problem of oxygen because theycontain oxygen scavenging molecules called leghaemoglobin. In nodules,leghaemoglobin may regulate oxygen in a similar way as hemoglobin doesin mammalian tissues. Nodules that are actively fixing nitrogen willappear reddish or pink, which can be evident from the exterior or whenthe nodule is cut open. In extreme cases the reddish color will extendinto the roots themselves. Tan colored nodules are not actively fixingbacteria and white, grey or green colored nodules are doing littlenitrogen fixing or could be dying.

To optimize legume crop yield it is important to maintain the propersoil fertility, high nodulation and high level of nitrogen fixingactivity are the keys to maintaining nitrogen levels in legumes.Depending on the particular legume and soil conditions, the plant mightobtain a small percentage or a majority of its nitrogen from fixation.Adding nitrogen fertilizer can be detrimental because some legumes don'trespond to nitrogen fertilizer. In other cases, because the nitrogenfixation process is energy intensive the plant may not expend energy fornodulation if it can absorb nitrogen directly from the added fertilizerusing less energy. This process uptakes less nitrogen than by fixationand yields can be compromised.

The legume is efficient in using the nitrogen that is fixed by itspartner bacteria. Almost all nitrogen that is fixed is used by theplant. Higher levels of nitrogen fixation translate to higher yield. Butthe higher rate of nodule formation does not always translate intohigher yield. Typically, only a small amount might leak to neighboringplants. Only when the plant dies does it return nitrogen to the soil andrelative to the amount of biomass of stems, leaves and roots that isturned into the soil.

Inoculation methods are numerous and revolve around delivery methods andspecific strains of bacteria to legume crops to increase nodulation. Akey element in activating the rhizobial nodulation (nod) genes ischemical signal that is sent by the host plant through its root hairs.Infection can happen only when root hairs are present. Flavonoidcompounds, such as LCO, are known to activate rhizobial nod genes. U.S.Pat. No. 7,250,068 describes methods of improving yield of a legume bytreating with the addition of lipo chitooligosaccharide (LCO), where,“an LCO which can increase the photosynthetic rate, and/or growth,and/or yield of a legume, in to acting as a trigger to initiate legumesymbiotic nitrogen fixation.”

U.S. Pat. No. 6,855,536 states that, “Unfortunately, for most of theUnited States, inoculation has been shown to be ineffective. Therefore,the inoculant industry remains relatively small (approximately $20-$30million per year.).”

Therefore, there is a need to provide a simple, broad based treatment toimprove nodulation of legumes and eliminate or reduce the need forinoculation.

SUMMARY OF THE INVENTION

Disclosed herein are methods of increasing, enhancing, or acceleratingroot nodulation in a plant, accelerating growth of nitrogen fixingbacteria in nodules of a plant, increasing protein content in a plant,increasing yield of a plant, improving water retention of a plant, orreducing water use of a plant, the method comprising identifying a plantin need of root nodulation, and applying to the plant a compositioncomprising a protein component comprising yeast stress proteinsresulting from subjecting a mixture obtained from the yeast fermentationto stress.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The treatment compositions disclosed herein are based on a fermentationproduct that is mixed with a surfactant, where the fermentation, basedon yeast, is either aerobic or anaerobic, and preferably incorporates amechanism to stress the yeast cells to yield essentially stressproteins. The proteins and surfactant form a complex to be termed theprotein/surfactant complex, or PSC. The process and compositions aredescribed in U.S. Pat. Nos. 7,476,529 and 7,658,848 and U.S. PatentApplication Nos. 20080167445, all of which are incorporated by referenceherein in their entirety, especially the passages describing thefermentation processes and the stress steps following the fermentationprocesses.

In certain embodiments, the protein component of the compositionsdisclosed herein are derived from the fermentation of yeast. In someembodiments, the fermentation is an aerobic fermentation, while in otherembodiments the fermentation is an anaerobic fermentation. In someembodiments, the protein systems disclosed herein are derived from anaerobic fermentation of Saccharomyces cerevisiae, which, when blendedwith surface active agents or surfactants, enhance multiple chemicalfunctions, at ambient conditions, or during and after exposure to theextreme conditions. The protein systems disclosed herein can also bederived from the fermentation of other yeast species, for example,kluyveromyces marxianus, kluyveromyces lactis, candida utilis,zygosaccharomyces, pichia, or hansanula.

After the aerobic fermentation process a fermentation mixture isobtained, which comprises the fermented yeast cells and the proteins andpeptides secreted therefrom. In some embodiments, the fermentationmixture can be subjected to additional stress, such as overheating,starvation, oxidative stress, or mechanical or chemical stress, toobtain a post-fermentation mixture. The post-fermentation stress causesadditional proteins to be expressed by the yeast cells and released intothe fermentation mixture to form the stress protein mixture. Theseadditional proteins are not normally present during a simplefermentation process. The additional proteins are known as “stressproteins,” and are sometimes referred to as “heat shock proteins”. Oncethe post-fermentation mixture is centrifuged, the resulting supernatantcomprises both the stress proteins and proteins normally obtained duringfermentation. The compositions described herein comprise stressproteins.

Several, rather low molecular weight proteins can be produced bySaccharomyces cerevisiae during fermentation as practiced by thosefamiliar in the art. These proteins appear when the yeast cells havebeen placed under stress conditions during or near the end of thefermentation process. Although referred to as “heat shock proteins,” thestress conditions can occur during periods of very low food to massconcentrations, or as the result of heat shock or pH shock conditions asdescribed in U.S. Pat. No. 6,033,875, Bussineau, et al., incorporated byreference herein in its entirety. In addition, chemical stress,oxidative stress, ultrasonic vibration and other stress conditions cancause the yeast to express the formation of heat shock proteins, moreaccurately termed, “stress proteins.”

Conditions for the post-fermentation procedures that produce the “heatshock proteins” are described in above-incorporated U.S. patents andpublications. As is clear from the passages in the '414 publication, andthe passages below, the regular fermentation steps do not generate heatshock proteins. Steps that generate heat shock proteins are administeredafter the fermentation step. It is necessary for the generation of heatshock proteins to cause shock to the fermented yeasts. This shockincludes, for example, rapid increase in the temperature, rapid changein the pH of the fermentation broth, rapid physical stress, and thelike.

As used herein, the term “protein component” refers to a mixture ofproteins that includes a number of proteins having a molecular weight ofbetween about 100 and about 450,000 daltons, and most preferably betweenabout 500 and about 50,000 daltons, and which, when combined with one ormore surfactants, enhances the surface-active properties of thesurfactants. In some embodiments, the protein component comprises amixture of multiple intracellular proteins and compounds, where at leasta portion of the mixture includes yeast polypeptides obtained fromfermenting yeast and yeast stress proteins resulting from subjecting amixture obtained from the yeast fermentation to stress. The “multipleintracellular proteins and compounds” includes proteins, small proteins,polypeptides, protein fragments, and the like, that are not normallyexpressed by yeast cells during the fermentation process. These proteinsand compounds are only expressed when the yeast cells are subjected tostress and shock following the fermentation process.

In a first example, the protein component comprises the supernatantrecovered from an aerobic yeast fermentation process. Yeast fermentationprocesses are generally known to those of skill in the art, and aredescribed, for example, in the chapter entitled “Baker's YeastProduction” in Nagodawithana T. W. and Reed G., Nutritional Requirementsof Commercially Important Microorganisms, Esteekay Associates,Milwaukee, Wis., pp 90-112 (1998), which is hereby incorporated byreference. Briefly, the aerobic yeast fermentation process is conductedwithin a reactor having aeration and agitation mechanisms, such asaeration tubes and/or mechanical agitators. The starting materials(e.g., liquid growth medium, yeast, a sugar or other nutrient sourcesuch as molasses, corn syrup, or soy beans, diastatic malt, and otheradditives) are added to the fermentation reactor and the fermentation isconducted as a batch process.

After fermentation, the fermentation product may be subjected toadditional procedures intended to increase the yield of the proteincomponent produced from the process. Several examples ofpost-fermentation procedures are described in more detail below. Otherprocesses for increasing yield of protein component from thefermentation process may be recognized by those of ordinary skill in theart.

The supernatant is obtained when the fermentation broth is centrifugedand the cellular debris is separated from liquid broth. While in someembodiments, as discussed above, the supernatant of the fermentationprocess is used in preparing the compositions described herein, in otherembodiments, the fermentation broth is used without any processing.Therefore, in these embodiments, the mixture used in preparing thecompositions described herein is the fermentation broth containingexcreted proteins and polypeptides and cellular debris, and wholeyeasts.

Saccharomyces cerevisiae is a preferred yeast starting material,although several other yeast strains may be useful to produce yeastferment materials used in the compositions and methods described herein.Additional yeast strains that may be used instead of or in addition toSaccharomyces cerevisiae include Kluyveromyces marxianus, Kluyveromyceslactis, Candida utilis (Torula yeast), Zygosaccharomyces, Pichiapastoris, and Hansanula polymorpha, and others known to those skilled inthe art.

In the first embodiment, Saccharomyces cerevisiae is grown under aerobicconditions familiar to those skilled in the art, using a sugar,preferably molasses or corn syrup, soy beans, or some other alternativematerial (generally known to one of skill in the art) as the primarynutrient source. Additional nutrients may include, but are not limitedto, diastatic malt, diammonium phosphate, magnesium sulfate, ammoniumsulfate zinc sulfate, and ammonia. The yeast is preferably propagatedunder continuous aeration and agitation between 30 to 35° C. and at a pHof 4.0 to 6.0. The process takes between 10 and 25 hours and ends whenthe fermentation broth contains between 4 and 8% dry yeast solids,(alternative fermentation procedures may yield up to 15-16% yeastsolids), which are then subjected to low food-to-mass stress conditionsfor 2 to 24 hours. Afterward, the yeast fermentation product iscentrifuged to remove the cells, cell walls, and cell fragments. It isworth noting that the yeast cells, cell walls, and cell fragments willalso contain a number of useful proteins suitable for inclusion in theprotein component described herein.

In an alternative embodiment, the yeast fermentation process is allowedto proceed until the desired level of yeast has been produced. Prior tocentrifugation, the yeast in the fermentation product is subjected toheat-stress conditions by increasing the heat to between 40 and 60° C.,for 2 to 24 hours, followed by cooling to less than 25° C. The yeastfermentation product is then centrifuged to remove the yeast cell debrisand the supernatant, which contains the protein component, is recovered.

In a further alternative embodiment, the fermentation process is allowedto proceed until the desired level of yeast has been produced. Prior tocentrifugation, the yeast in the fermentation product is subjected tophysical disruption of the yeast cell walls through the use of a FrenchPress, ball mill, high-pressure homogenization, or other mechanical orchemical means familiar to those skilled in the art, to aid the releaseof intracellular, polypeptides and other intracellular materials. It ispreferable to conduct the cell disruption process following a heatshock, pH shock, or autolysis stage. The fermentation product is thencentrifuged to remove the yeast cell debris and the supernatant isrecovered.

In a still further alternative embodiment, the fermentation process isallowed to proceed until the desired level of yeast has been produced.Following the fermentation process, the yeast cells are separated out bycentrifugation. The yeast cells are then partially lysed by adding 2.5%to 10% of a surfactant to the separated yeast cell suspension (10%-20%solids). In order to diminish the protease activity in the yeast cells,1 mM EDTA is added to the mixture. The cell suspension and surfactantsare gently agitated at a temperature of about 25° to about 35° C. forapproximately one hour to cause partial lysis of the yeast cells. Celllysis leads to an increased release of intracellular proteins and otherintracellular materials. After the partial lysis, the partially lysedcell suspension is blended back into the ferment and cellular solids areagain removed by centrifugation. The supernatant, containing the proteincomponent, is then recovered.

In a still further alternative embodiment, fresh live Saccharomycescerevisiae is added to a jacketed reaction vessel containingmethanol-denatured alcohol. The mixture is gently agitated and heatedfor two hours at 60° C. The hot slurry is filtered and the filtrate istreated with charcoal and stirred for 1 hour at ambient temperature, andfiltered. The alcohol is removed under vacuum and the filtrate isfurther concentrated to yield an aqueous solution containing the proteincomponent.

The compositions described herein include one or more surfactants at awide range of concentration levels. Some examples of surfactants thatare suitable for use in the detergent compositions described hereininclude the following:

Anionic: Sodium linear alkylbenzene sulphonate (LABS); sodium laurylsulphate; sodium lauryl ether sulphates; petroleum sulphonates;linosulphonates; naphthalene sulphonates, branched alkylbenzenesulphonates; linear alkylbenzene sulphonates; alcohol sulphates; POand/or PO/EO sulfated alcohols.

Cationic: Stearalkonium chloride; benzalkonium chloride; quaternaryammonium compounds; amine compounds.

Non-ionic: Dodecyl dimethylamine oxide; coco diethanol-amide alcoholethoxylates; linear primary alcohol polyethoxylate; alkylphenolethoxylates; alcohol ethoxylates;

EO/PO polyol block polymers; polyethylene glycol esters; fatty acidalkanolamides.

Amphoteric: Cocoamphocarboxyglycinate; cocamidopropylbetaine; betaines;imidazolines.

In addition to those listed above, suitable nonionic surfactants includealkanolamides, amine oxides, block polymers, ethoxylated primary andsecondary alcohols, ethoxylated alkylphenols, ethoxylated fatty esters,sorbitan derivatives, glycerol esters, propoxylated and ethoxylatedfatty acids, alcohols, and alkyl phenols, alkyl glucoside glycol esters,polymeric polysaccharides, sulfates and sulfonates of ethoxylatedalkylphenols, and polymeric surfactants. Suitable anionic surfactantsinclude ethoxylated amines and/or amides, sulfosuccinates andderivatives, sulfates of ethoxylated alcohols, sulfates of alcohols,sulfonates and sulfonic acid derivatives, phosphate esters, andpolymeric surfactants. Suitable amphoteric surfactants include betainederivatives. Suitable cationic surfactants—include amine surfactants.Those skilled in the art will recognize that other and furthersurfactants are potentially useful in the compositions depending on theparticular detergent application.

Preferred anionic surfactants used in some detergent compositionsinclude CalFoam® ES 603, a sodium alcohol ether sulfate surfactantmanufactured by Pilot Chemicals Co., and Steol® CS 460, a sodium salt ofan alkyl ether sulfate manufactured by Stepan Company. Preferrednonionic surfactants include Neodol® 25-7 or Neodol® 25-9, which areC12-C15 linear primary alcohol ethoxylates manufactured by ShellChemical Co., and Genapol® 26 L-60, which is a C12-C16 natural linearalcohol ethoxylated to 60E C cloud point (approx. 7.3 mol), manufacturedby Hoechst Celanese Corp.

Several of the known surfactants are non-petroleum based. For example,several surfactants are derived from naturally occurring sources, suchas vegetable sources (coconuts, palm, castor beans, etc.). Thesenaturally derived surfactants may offer additional benefits such asbiodegradability.

One of the features of the PSC is the ability to accelerate uptake ofnutrients and accelerate metabolic processes of aerobic bacteria basedon a mechanism called uncoupling of oxidative phosphorylation. And ithas been shown that the uncoupling effect and its benefits are observedat low temperature as well as at ambient temperatures. An effect of thisfeature is to limit the amount of biomass being formed, including theamount of biofilm being developed, typically based on polysaccharides.The uncoupling effect uncouples the microbe's ability to form complexproteins. Nitrogenase is a complex protein and it would be expected thatthe level of nitrogenase would be reduced with the uncoupling factor ofthe PSC. To protect the needed nitrogenase system in the nodulationprocess when aerobic bacteria are present, species like Azotobacter andRhizosium (The Microbial World: The Nirogen cycle and Nitrogen fixation,Jim Deacon, University of Edinburgh) produce large amounts ofextracellular polysaccharide to limit the rate of diffusion of oxygeninto cells. The PSC treatment would appear to be detrimental to thenitrogenase based on these phenomena. The results of the tests, however,show otherwise.

The Rhizobia bacteria infect a plant's roots through its root hairs. Wehave observed that PSC treated plants had a substantially greater amountof fine root hair.

It is a hypothesis, but not a limitation of the current invention, thatthe mechanism for the enhanced nodulation when treated by the PSC is dueto the following factors. The increased uptake of nutrient by bacteriain soil treated by the PSC accelerates the growth rate of appropriatenitrogen fixing bacteria in the plant and the plant responds in kind byincreasing the amount of leghaemoglobin it produces in the nodules. Theeffect is noted by the intensity of the reddish color observed in thetreated nodules, which can extend up into the roots. Further, since thePSC has been shown to accelerate the growth of fine root hairs, thenthis is believed to be an additional embodiment of the current inventionthat improves nodulation of legumes. The increased uptake of nutrient,as in nitrogen and Nod factors, is hypothesized to be a factor in thehigher rate of nitrogen fixation, which is noted by the reddish color ofthe nodules.

Thus, in one aspect, disclosed herein are methods of increasing,enhancing, or accelerating root nodulation in a plant, the methodcomprising identifying a plant in need of root nodulation, and applyingto the plant a composition comprising a protein component comprisingyeast stress proteins resulting from subjecting a mixture obtained fromthe yeast fermentation to stress.

In another aspect, disclosed herein are methods of accelerating growthof nitrogen fixing bacteria in nodules of a legume, the methodcomprising identifying a legume in need thereof, and applying to thelegume a composition comprising a protein component comprising yeaststress proteins resulting from subjecting a mixture obtained from theyeast fermentation to stress.

In another aspect, disclosed herein are methods of increasing proteincontent in a legume, the method comprising identifying a legume in needthereof, and applying to the legume a composition comprising a proteincomponent comprising yeast stress proteins resulting from subjecting amixture obtained from the yeast fermentation to stress.

In another aspect, disclosed herein are methods of increasing yield of alegume, the method comprising identifying a legume in need thereof, andapplying to the legume a composition comprising a protein componentcomprising yeast stress proteins resulting from subjecting a mixtureobtained from the yeast fermentation to stress.

In some embodiments of the above methods, the protein component isobtained through the processes described above. In some embodiments, theplant in need of such methods is a plant being used to increase thenitrogen content of soil during crop rotation, or a plant required toprovide higher yield, or higher nutrition content, or a plant requiredto have reduced water use.

In some embodiments of the above methods, the composition is applied tothe soil near the plant. In some of these embodiments, the compositionis applied through irrigation, which can be spray irrigation or dripirrigation. In certain embodiments, the composition is applied withevery watering cycle or, in other embodiments, on an intermittent basis.

In some embodiments of the above methods, the protein component is fromaerobic fermentation of yeast. In some of these embodiments, the proteincomponent comprises proteins obtained from exposing a product obtainedfrom the fermentation of yeast to additional procedures that increasethe yield of proteins produced from the fermentation. In certainembodiments, the stress is selected from the group consisting of heatshock of the fermentation product, physical and/or chemical disruptionof the yeast cells to release additional polypeptides, and lysing of theyeast cells. In further embodiments, the stress comprises exposing aproduct obtained from the fermentation of yeast to heat shockconditions. In some embodiments, the stress comprises physicallydisrupting the yeast after the fermentation of the yeast, while in otherembodiments, the stress comprises chemically disrupting the yeast afterthe fermentation of the yeast. In some embodiments, the stress compriseslysing the yeast after the fermentation of the yeast.

In some embodiments of the above methods, the methods further comprisemixing the protein component with additional nutrients prior to theapplication to the plant. The additional nutrients include, but are notlimited to, fertilizers, sources of phosphate, minerals, herbicides, andinsecticides.

In some embodiments of the above methods, the composition furthercomprises one or more of an anionic surfactant, a non-ionic surfactant,a cationic surfactant, and amphoteric surfactant, as described elsewhereherein.

In some embodiments of the above methods, the yeast is selected from thegroup consisting of Saccharomyces cerevisiae, Kluyveromyces marxianus,Kluyveromyces lactis, Candida utilis (Torula yeast), Zygosaccharomyces,Pichia pastoris, and Hansanula polymorpha.

In some embodiments of the above methods, the plant is a legume. Incertain embodiments, the legume is selected from the group consisting ofalfalfa, clover, peas, beans, lentils, lupins, mesquite, carob, soy,peanuts, locust trees (Gleditsia or Robinia), wisteria, and the Kentuckycoffeetree (Gymnocladus dioicus).

In other embodiments of the above methods, a volume of soil is premixedwith the above composition to form a mixture, and then the mixture isapplied to the plant. Thus, in another aspect, disclosed herein is asoil mixture comprising soil and a composition comprising a proteincomponent comprising yeast stress proteins resulting from subjecting amixture obtained from the yeast fermentation to stress, as describedabove.

In another aspect, disclosed herein are methods of improving waterretention of a legume, reducing water use of a legume, accelerating rootnodulation in a legume, accelerating nitrogen fixation by a legume,accelerating growth of nitrogen fixing bacteria in nodules of a legume,increasing protein content in a legume, or increasing yield of a legume,the method comprising identifying a legume in need thereof, and applyingto the legume a soil mixture as described above.

In some embodiments of the above methods, the methods further comprisesinoculating the soil with bacteria prior to the application of thecomposition. In other embodiments, the methods are practiced withoutinoculation of the soil with any bacteria.

In yet another aspect, disclosed herein are methods of increasing thetolerance of a legume to colder climates, the method comprisingidentifying a legume in need thereof, and applying to the legume aprotein component comprising yeast stress proteins resulting fromsubjecting a mixture obtained from the yeast fermentation to stress, asdescribed above.

Example 1

A fermentation mixture derived from the fermentation of Saccharomycescerevisieae in which the yeast cells are stressed by raising thetemperature to at least 35° C. for at least two hours, then cooling to<30° C. centrifugation. Upon removal of the yeast cells bycentrifugation the pH is adjusted to 4.0 and sodium benzoate and 21.1%propylene glycol is incorporated to provide stability.

PSC Linear Primary Alcohol (C12-C15), 7 mole Ethoxylate 7.5% SodiumLauryl Ether (3 mole) Sulfate (60%) 2.5% Stabilized Fermentation Mixture 23% Water  67% TOTAL 100% 

Host plants: Peas, Pisum sativum. Two seeds were sown in 6 inch diampots (approx. 3500 ml volume) filled with UC Mix II (Matkin and Chandler1957). Soil Mix II is formulated with plaster sand, bark, peat moss,Dolomite, limestone flour, triple super phosphate, potassium nitrate,muriate of potash, ferrous sulfate, copper sulfate, magnesium sulfate,zinc sulfate, and manganese sulfate. Once sown, the pots were watered ona daily basis until the plant was visible above ground. The resultingplants were culled to one plant per pot at the cotyledon (seed leaf)stage. Treatment applications commenced following culling.

Host Plant Care: Plants were placed on raised greenhouse benches forstudy. The plants were fertilized with Miracle-Gro (with minors)general-purpose fertilizer at 200 ppm nitrogen. Fertigation protocol wassuch that each plant in the experiment received the same amount offertilizer throughout the experiment.

Applications: A control treatment of water only was used in thisexperiment. Protein Surfactant Combination PSC was applied by hand at 75ppm three times a week. Product was added to water at the appropriateconcentration such that each plant received 90 ml of solution at everyapplication. Water at 90 ml was added to each control plant whentreatment applications were performed.

Experimental Design: There were eight plants that were treated and thenumber of treatments of control pots that had no PSC treatment.

Sampling: A destructive sample was taken at 30 and 60 days aftertreatment initiation. Root weight and rhizome production weredetermined.

Results

Mean (grams±SE) dry weight of roots and the number and dry weight ofrhizomes of sweet peas treated with selected protein surfactantcombinations.

Mean root Mean no. of Mean dry weight Treatment dry weight¹ rhizomes² ofrhizomes¹ PSC 2.16 ± 0.15a 49.2 ± 12.7a 0.086 ± 0.014 Control 1.93 ±0.14a 31.6 ± 12.6a 0.040 ± 0.014 ¹Means followed by different lettersare significantly different, LSD (p = 0.05) ²Means are significantlydifferent at p = 0.01, ChiSq = 5.11, df = 2, P = 0.0775.

Discussion

The PSC nodules were more than twice the weight of the Control and therewere 56% more nodules than the Control. Further, the PSC treated noduleswere reddish in color, indicating a high level of nitrogen fixation. TheControl nodules had a brownish color, indicating little nitrogenfixation. This suggest a higher protein content and higher crop yield.

1. A method of increasing, enhancing, or accelerating root nodulation ina plant, accelerating growth of nitrogen fixing bacteria in nodules of aplant, increasing protein content in a plant, increasing yield of aplant, improving water retention of a plant, or reducing water use of aplant, the method comprising identifying a plant in need of rootnodulation, and applying to the plant a composition comprising a proteincomponent comprising yeast stress proteins resulting from subjecting amixture obtained from the yeast fermentation to stress.
 2. The method ofclaim 1, wherein the composition is applied to the soil near the plant.3. The method of claim 1, wherein the composition is applied throughirrigation.
 4. The method of claim 3, wherein the irrigation is sprayirrigation or drip irrigation.
 5. The method of claim 1, wherein thecomposition is applied with every watering cycle or intermittent basis.6. The method of claim 1, wherein the protein component is from aerobicfermentation of yeast.
 7. The method of claim 1, wherein the proteincomponent comprises proteins obtained from exposing a product obtainedfrom the fermentation of yeast to additional procedures that increasethe yield of proteins produced from the fermentation.
 8. The method ofclaim 1, wherein the stress is selected from the group consisting ofheat shock of the fermentation product, physical and/or chemicaldisruption of the yeast cells to release additional polypeptides, andlysing of the yeast cells.
 9. The method of claim 1, wherein the stresscomprises exposing a product obtained from the fermentation of yeast toheat shock conditions.
 10. The method of claim 1, wherein the stresscomprises physically or chemically disrupting the yeast after thefermentation of the yeast.
 11. The method of claim 1, wherein the stresscomprises lysing the yeast after the fermentation of the yeast.
 12. Themethod of claim 1, further comprising mixing the protein component withadditional nutrients prior to the application to the plant.
 13. Themethod of claim 1, wherein the composition further comprises one or moreof an anionic surfactant, a non-ionic surfactant, a cationic surfactant,and amphoteric surfactant.
 14. The method of claim 1, wherein the yeastis selected from the group consisting of Saccharomyces cerevisiae,Kluyveromyces marxianus, Kluyveromyces lactis, Candida utilis (Torulayeast), Zygosaccharomyces, Pichia pastoris, and Hansanula polymorpha.15. The method of claim 1, wherein the plant is a legume.
 16. The methodof claim 12, wherein the legume is selected from the group consisting ofalfalfa, clover, peas, beans, lentils, lupins, mesquite, carob, soy,peanuts, locust trees (Gleditsia or Robinia), wisteria, and the Kentuckycoffeetree (Gymnocladus dioicus).
 17. A soil mixture comprising soil anda composition comprising a protein component comprising yeast stressproteins resulting from subjecting a mixture obtained from the yeastfermentation to stress.
 18. A method of improving water retention of alegume, reducing water use of a legume, accelerating root nodulation ina legume, accelerating nitrogen fixation by a legume, acceleratinggrowth of nitrogen fixing bacteria in nodules of a legume, increasingprotein content in a legume, or increasing yield of a legume, the methodcomprising: identifying a legume in need thereof, and applying to thelegume a soil mixture of claim 17.